Cloning and Functional Identification of Monoterpene Synthase Gene NtTPS2 in Tobacco

doi:10.13560/j.cnki.biotech.bull.1985.2020-1492

Abstract

Abstract:

Terpenoids constitute the most abundant and diverse family in structure and function of the secondary metabolites of plants. They play important roles in the regulation of plant growth and development,stress responses and interactions with environmental biotic and abiotic factors. To study the biosynthesis of terpenoids in tobacco（Nicotiana tabacum）,a monoterpene synthase was cloned from N. tabacum,named as NtTPS2. NtTPS2 contained an open reading frame（ORF）of 1 854 bp,encoding a protein of 617 amino acids with a N-terminal signal peptide and conserved DDxxD and NSE/DTE domains. Phylogenetic analysis revealed that NtTPS2 was in the TPS-II clade sharing high amino acid sequence identities with monoterpene synthase genes from petunia（Petunia hybrida）,lemon（Citrus limon）and citrus（Citrus reticulata Blanco）. NtTPS2 was located in the chloroplast in plant cells. Real-time PCR analysis showed that NtTPS2 expressed abundantly in the roots and pistils,and responded to the treatments of low temperature,high salt,drought and ABA. When NtTPS2 was co-expressed in Escherichia coli with mevalonate pathway and geranyl diphosphate synthase,the formation of monoterpene geraniol and its isomer nerol was detected. These results suggest that NtTPS2 plays a key role in the biosynthesis and metabolism of monoterpene in tobacco.

Key words: Nicotiana tabacum, NtTPS2, monoterpene synthase, geraniol, nerol

LIU Shao-hua, ZHAO Xi-sheng, YANG Qing, YANG Chang-qing, PAN Xu-hao, ZHANG Jian-hui, YANG Ai-guo, LI Yi-ting. Cloning and Functional Identification of Monoterpene Synthase Gene NtTPS2 in Tobacco[J]. Biotechnology Bulletin, 2021, 37(9): 132-141.

Figures/Tables 9

Table 1 Primers for NtTPS2 gene cloning,vector construction and qPCR

引物名称 Primer	引物序列Sequence（5'-3'）
NtTPS2-F	ATGGCCACCTCCATAAGACCTGCAA
NtTPS2-R	TTATAGGGATGGATTGGGAGTCAAT
NtTPS2∷gfp-F	CAGTGGTCTCACAACATGGCCACCTCCATAAGACC
NtTPS2∷gfp-R	CAGTGGTCTCATACATAGGGATGGATTGGGAGTCA
NtTPS2-pYJM26-F	GAAGATCTATGGCCACCTCCATAAGACCTGCAA
NtTPS2-pYJM26-R	CCCTCGAGTTATAGGGATGGATTGGGAGTCAAT
Actin-F	TTCCGATGCCCTGAAGTCCT
Actin-R	TCTGCCTTTGCAATCCACAT
NtTPS2-qF	AAATGCCAAACGCTAATCCT
NtTPS2-qR	TTCCACCACCTTGATACATCTC

Fig.1 PCR amplification of NtTPS2 M：RB 2 000 DNA molecular marker;1：PCR amplification product

Fig.2 Prediction of the conserved domain of NtTPS2 protein A：Terpene synthase,N-terminal domain;B：isoprenoid synthase domain,terpene synthase,metal-binding domain

Fig.3 Multiple sequence alignment of NtTPS2 and other plant terpene synthase genes The red boxes are marked as conserved domains of terpene synthase “DDxxD” and “（N,D）D（L,I,V）X（S,T）XXXE”. AcNES：Actinidia chinensis nerol synthase（AER36088.1）;MsLIS：Mentha spicata limonene synthase（AAC37366.1）;ObZIS：Ocimum basilicum α-pinene synthase（AAV63788.1）;AaLIS：Artemisia annua linalool synthase（AAF13356.1）;VvGES：Vitis vinifera geraniol synthase（NP_001267895.1）

Fig.4 Phylogenetic tree analysis of NtTPS2 and terpene synthase of other species

Fig.5 Subcellular localization of NtTPS2

Fig.6 Tissue expression pattern analysis of NtTPS2 Different letters indicate significant differences at the P<0.05 level. The same below

Fig.7 Expression analysis of NtTPS2 under different stresses

Fig.8 Analysis of metabolites of NtTPS2 A：GC-MS analyses of mixed standard products of geraniol and nerol;B：GC-MS analyses of geraniol and nerol synthesized by NtTPS2 engineering bacteria;C：GC-MS analyses of products synthesized by control engineering bacteria;D：mass spectrum of nerol standard;E：mass spectrum of geraniol standard;F：mass spectrum of nerol synthesized by NtTPS2 engineering bacteria;G.：mass spectrum of geraniol synthesized by NtTPS2 engineering bacteria

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